Methods for disinfecting or sterilizing articles

ABSTRACT

Disclosed herein are materials and methods for reducing, preventing, or eliminating the biological contamination of surfaces in spaces that are reasonably partitioned, defined or contained. A reagent that includes peroxides or molecules that includes peroxide bonds are contacted with at least one surface of an article. Some of these methods include disinfection within an apparatus and may include the step of volatilizing the peroxide bond containing agent, through, for example, vapour pressure and vaporization or evaporation effects, the addition of volatilization aids, or passive or assisted diffusion at modest temperatures and relatively long exposure times measured in some embodiments on the order of hours, days, or months. In some embodiments a liquid that includes a peroxide moiety is added (for example, during manufacture or subsequently) to a package, container, or any other reasonably partitioned or contained volume and allowed to migrate within the confined space for a period of time sufficient to disinfect the inner surfaces of the container and articles it contains.

FIELD OF THE INVENTION

This invention relates generally to materials and methods forsterilizing or disinfecting a variety of devices, articles andmaterials; these methods can be practiced in situ and include methodsfor eliminating or reducing the biological contamination of the interiorvolume and surfaces of containers or structures.

BACKGROUND

Microorganisms, such as bacteria, fungi and the like are virtuallyubiquitous in the biosphere and while most are harmless, many have thecapacity to infect humans, plants and animals while still others cancause spoilage of valuable foodstuffs and other materials. Accordingly,there exists a pressing, ongoing need to control, if not eliminate,unwanted microorganisms from a wide variety of surfaces. One of the mostcommon methods of disinfecting or sterilizing surfaces is the use ofheat, especially wet heat in the form of high pressure steam.Unfortunately, these methods do not work well with materials that areheat and/or water labile and oftentimes it is difficult if notimpossible to employ these methods in situ.

For heat and/or moisture sensitive materials, alternatives to dry heatand/or steam must be used to control the biological contamination ofthese materials. Some of these alternatives include, but are not limitedto, irradiation, exposure to toxic gases (for example, ethylene oxide,chlorine dioxide, or ozone), ultraviolet radiation, or treatment withchemical bactericides, fungicides and the like. Many of these methodsare difficult to use with various articles of manufacture so thereremains a need for new materials and methods for controlling microbialcontamination on surfaces and articles. Some aspects of the instantinvention address these needs.

SUMMARY

Some embodiments of the invention provide methods for disinfectingand/or sterilizing the interior volume and surfaces of sealed containersor structures using peroxides and molecules that include peroxide bonds.Some embodiments provide methods for disinfecting and/or sterilizingsurfaces which leave behind small, generally recognized as safe (GRAS)levels of chemical residuals.

Some embodiments are methods performed using articles such as packages,containers, bags, pouches, packaging, wrappings, tubs, tins, bottles,buckets, and formed structures, or any other article; preferably thosearticles having a reasonably partitioned or defined space or volumewithin which a reagent that may include a peroxide is inoculated,sprayed, or otherwise placed in contact with at least one surface of thearticle. Some of these methods include disinfection within an apparatusand may include the step of volatilization of the peroxide bondcontaining agent, through, for example, vapor pressure and evaporativeeffects, the use of volatilization aids, or passive or assisteddiffusion at modest temperatures and providing relatively long exposuretimes, measured in some embodiments on the order of hours, days, ormonths. In some embodiments a liquid that includes a peroxide moiety isadded (for example, during manufacture or subsequently) to a package,container, or any other reasonably partitioned volume and allowed tomigrate within the defined space for a period of time sufficient todisinfect the inner surfaces of the container.

Some embodiments of the present invention also use as the active agentperformic acid (generated by the mixture of water, formic acid, andhydrogen peroxide with or without volatilization aids) for the controlor elimination of microorganisms from surfaces.

Some embodiments include methods for controlling contamination within apartitioned or otherwise defined space, comprising the steps ofcontacting surfaces within a partitioned or otherwise defined space witha chemical in the vapor phase. In some embodiments the chemical in thevapor phase is selected from the group consisting of: peroxides,peracids, or moieties that include at least one peroxide bond. In someembodiments the surfaces are exposed to the chemical in its vapor phasefor at least 5 minutes at a temperature below 50 C. In some embodimentsthe partitioned or otherwise defined space includes at least one articlethat is not an integral part of the partitioned or defined space orvolume.

In some embodiments the partitioned of otherwise defined space is acontainer. In some embodiments the container is selected from groupconsisting of: packaging, containers, bags, pouches, cans, tins,buckets, tubs, bottles, ampoules, barrels, sleeves, capsules, boxes,other formed packages or structures, and the like. In some embodimentsthe containers are flexible. And in other embodiments the containers arerigid. In some embodiments the container may include at least oneaccessory or feature. In some embodiments the accessory or feature isselected from the group consisting of: fitments, valves, bungs, lids,vents, ports, spouts, drains, covers and caps, seals, or membrane seals.

In some embodiments surfaces of or within a partitioned or defined spaceor volume are incubated with a peroxide, peracid, or a mixture ofcompounds, one or more of which includes a peroxide bond for longer thanabout five minutes. In some embodiments the temperature the incubationstep occurs at a temperature equal to or less than about 50 C. In someembodiments a peroxide concentration equal to or greater than about 2%is added to or used in a partitioned or defined space to form a peracidor a mixture of compounds one or more of which includes a peroxide bond.

In some embodiments the peroxide mixture added to or used in thepartitioned or defined space includes formic acid (methanoic acid) orperformic acid (permethanoic acid) either of which may be added to orpresent at a concentration greater than or equal to about 2%. In someembodiments the peroxide mixture added to or used in the partitioned ordefined space includes peracetic acid (ethanoic acid) or peracetic acid(perethanoic acid), or propionoic acid (propanoic acid) or propionoicperacid (propanoic peracid), or a mixture of these with or without theaddition of formic acid, any of or the combination of which is added orpresent at a concentration greater than or equal to 2%. In someembodiments the peroxide mixture added to or used in the partitioned ordefined space includes at the time of addition, or subsequently evolvesto form, a mixture that is generally recognized as safe for consumptionor other usage.

Still other embodiments include methods for reducing the number ofbiological contaminants in a partitioned or otherwise defined space orvolume, comprising the steps of: mixing formic acid, hydrogen peroxideand water with or without the addition of other compounds; wherein themixture produces performic acid in situ; and contacting at least onesurface with said performic acid formed in situ wherein the surface iswithin a partitioned or otherwise defined space or volume. In some ofthese embodiments the space or volume is the interior of a container. Insome embodiments the container is selected from the group consisting of;packaging, bags, pouches, cans, tins, boxes, buckets, tubs, bottles,ampoules, barrels, sleeves, formed packages or structures or the like.In some embodiments the partitioned or otherwise defined space or volumeincludes at least one article and the article is present in the space orvolume at the same time as a microbiocidal concentration of peracid orperoxide bond containing vapors. In some embodiments the interior of thecontainer includes at least one article inside the container at the sametime as a microbiocidal concentration of peracid or peroxide bondcontaining vapors.

Still other embodiments include systems for controlling contaminationwithin a partitioned or otherwise defined space or volume comprising: avapor that includes a microbiocidal concentration of peracid or peroxidebonds within a partitioned or otherwise defined space or volume, whereinthe concentration of vapors is such that at least about 5 minutes ofcontact between the vapor and an interior surface of the space or volumeis sufficient to inactivate at least about 90 percent of the microbeswithin the space or volume. In some embodiments the vapor is formed by amixture that includes peracid or peroxide formic acid (methanoic acid)or performic acid (permethanoic acid) either of which is added orpresent at a concentration greater than or equal to about 2%. In someembodiments the system is used to treat the interior of a containerwhich may or may not house an article that is not an integral part ofthe container.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Photograph of the inner surface of a fitment cap showing thedried bacterial spore inoculum. The 10 microliter inoculum dries toleave behind a circular, thick cream colored spore mass.

FIG. 1B. Similar to 1A, a photograph of the inner surface of a fitmentcap showing the dried bacterial spore inoculum.

FIG. 2. The 10 microliter spore inoculum dried onto the fitment shown inFIG. 1 viewed at about 25 times magnification using a stereomicroscope.

FIG. 3. Two 57.9 gallon drum bags. For testing, some bags were incubatedin a flat position (bottom bag), while other bags were treated in afolded configuration (lower right hand corner) after depositing adisinfectant mixture in the bottom of the bag in order to demonstratethe ability of the disinfecting mixture to migrate past the folds.

FIG. 4. Graph of the change in weight over time of polyolefin bagscontaining water or a disinfecting mixture. The weight loss is due tomigration of water and/or components of the disinfection mixture throughthe walls of the bag.

DESCRIPTION

For the purposes of promoting an understanding of the principles of thenovel technology, reference will now be made to the preferredembodiments thereof, and specific language will be used to describe thesame. It will nevertheless be understood that no limitation of the scopeof the novel technology is thereby intended; such alterations,modifications, and further applications of the principles of the noveltechnology being contemplated as would normally occur to one skilled inthe art to which the novel technology relates are within the scope ofthis disclosure and what it claims.

Unless specifically stated or clearly implied the term ‘about’ as usedherein refers to a range of values from less than to greater than 10percent of the stated value. For example, about 1 refers to the range ofvalues from 0.9 to 1.1

Multiple methods and features of the present invention are novel inrelation to current practice. Though peroxide and peracids are commonlyused for disinfection and sterilization of containers and packaging(s),they are most often employed so as to produce rapid activity for on-linedisinfection at relatively short exposure times. The active agents aregenerally applied by immersion, spraying, filling, or other methodsdesigned to distribute the active agents rapidly and throughout or overthe product to be disinfected or sterilized. The disinfection agents aremost often used at high concentration and volume, and/or with theaddition of a stimulant such as heat, ultraviolet light, a plasma richenvironment, or the like in order to activate the oxidative activity ofthe peroxide-moiety and accelerate its decay and attendant release ofits oxidative capacity.

Contrary to currently used commercial methods the present invention usesrelatively small volumes of disinfecting reagents, and relatively longtimes of exposure (for example, hours or days) to produce disinfectionor sterilization while yielding low levels of residual compounds. Theactive agents employed in the present invention are distributedthroughout the treated product by evaporation and other volatilizationmeans followed by passive or facilitated diffusion.

Some embodiments of the instant invention generate performic acid insitu. In some embodiments performic acid mixtures are produced bycombining water, formic acid, and hydrogen peroxide inside of the spacethat is to be disinfected. The utility of this approach may be furtherextended by partitioning or confining the space so as to allow theconcentration of performic acid or vapors from performic acid toaccumulate in the given space. Given enough time and the necessaryamount of performic acid in a given substantially confined or otherwisepartitioned space, this reagent can be used for the in situsterilization of surfaces in a partitioned space or volume.

Peroxide or peracid based reagents used for disinfecting or sterilizingsurfaces are usually used in combination with accelerators of peroxidedecomposition such as heat, ultraviolet light, plasmas, or otheractivators which speed-up the formation of hydroxyl radicals. Incontrast, many of the embodiments of the present invention can bepracticed without the need for such accelerants of peroxidedecomposition. These methods are especially useful for the disinfectionand/or or sterilization of sealed containers, such as packages,containers, bags, pouches, tanks, tubs, bottles, buckets, sleeves orother formed structures, using a small volume of hydrogen peroxide oranother peracid agent, in conjunction with long contact time. Theperoxide bond rich vapor migrates within the container and producesmicrobial disinfection on the surfaces and the volume within thecontainer through oxidative effects acting over a relatively long timeof exposure (for example, hours or days rather than a few seconds orminutes) at ambient or modestly elevated temperatures.

Some embodiments of the current invention are especially well suited forthe sterilization of bags for use in bag-in-box (BIB) applicationswithin the food and beverage industry. Currently, bags for BIBapplications are manufactured at one site and then the bag/fitmentcombination is sterilized at a second site using irradiation.Sterilization by irradiation in this process usually requires that thebags be transported to a distant irradiation facility, inserted into theirradiation processing queue, and transported back to the originator forsale and distribution following irradiation sterilization. This processis expensive due to the required shipping and processing (theirradiation process itself is relatively expensive in part because ofthe high capital costs associated with the facility and shielding of theradiation source), and time consuming (typically, the minimum timerequired for the round trip is one to two weeks or more).

In contrast, invention methods discussed herein can be used virtuallyanywhere. In some embodiments of the invention a small volume of areagent that may include a peroxide or peracid mixture is employed. Thereagent may be sprayed, painted, or otherwise dispensed into a bag, or aportion of the bag, or the neck of a fitment that opens into the bagbefore, or during, the attachment of the fitment to the bag.Volatilization of the active compounds within the bag disinfects theinner volume and surfaces of the bag including surfaces of the bag thatare not in direct contact with the liquid or solid form of the peroxidecontaining reagent. In some embodiments, the active disinfection mixturemay be added to a fitment containing a membrane seal, and/or to a bag towhich the fitment is subsequently affixed. By controlling the volume ofdisinfectant employed and/or continued disintegration of the activechemistries, it is usually possible to generate harmless and generallyrecognized as safe residuals and residual levels in the bag at the timeof filling. Accordingly, aspects of the present invention providerelatively rapid and inexpensive methods (relative to the transportationand processing times and costs generally attendant to conventionalradiation processing) for disinfecting or sterilizing surfaces inconfined spaces such as bags. These methods are readily adaptable forBIB applications.

In still other embodiments these methods are used to sterilize ordisinfect medical products or devices packaged for use at a later time,examples of such products, include, but are not limited to scalpels orscalpel blades, luers, shunts, catheters, sutures, and implants packagedin a wrap or container eventually sealed to exclude re-contamination bymicroorganisms. The inclusion of a small volume of disinfectant(according to some embodiments of the present invention) before sealingof the package provides a reagent for disinfecting or sterilizing theinterior of the package and the surfaces of any article(s) within thepackage. These inventive methods enable the manufacturer to avoid costlyradiation or other energy intensive methods of sterilization ordisinfection, such as use of a retort.

EXPERIMENTS AND EXEMPLARY RESULTS Experiment 1

About two or three droplets of 3% hydrogen peroxide were droppered intoa 4 ounce PET bottle before capping; the peroxide was omitted from twobottles which were used as control samples. Before sealing, the insidesurface of the bottle cap (liner removed) or its foam liner had beeninoculated with a 10 μL droplet of Bacillus atrophaeus ATCC 9372 sporesdried down onto the cap or liner surface. Bacillus atrophaeus ATCC 9372spores are known to be of relatively high resistance to peroxidedisinfection and are a recommended (for example, the U.S. Pharmacopeia)resistant organism for disinfection studies using chemicals (such asperoxide or ethylene oxide) or dry heat.

After standing overnight (15 hours) at 35 C the bottles were opened andthe inoculated areas (of both untreated, control bottles and bottles towhich hydrogen peroxide had been added) were swabbed to sample the levelof recoverable viable spores. The results obtained are shown in Table 1below. The level of spores recoverable from bottles to which 3 drops of3% hydrogen peroxide had been added was less (reductions >90% or >1logarithm lower) than that from control bottles to which no hydrogenperoxide had been added. Thus, the hydrogen peroxide mixture used inthis experiment did reduce the level of recoverable viable spores andshow some disinfection capability, however, the level of efficacy wassmall and insufficient to prove useful for the purposes ofsterilization.

TABLE 1 The recovery of Bacillus atrophaeus spores from 4 oz. PETbottles incubated with (3 Drops, Overnight) or without (None, RecoveryControl) the addition of 3% hydrogen peroxide. Site of CFU Log LogInoculation Treatment Recovered Recovery Reduction Liner None (Recovery2300000 6.4 NA Control) Cap None (Recovery 6600000 6.8 NA Control) Liner3 Drops, 830000 6 0.4 Overnight Cap 3 Drops, 425000 5.6 1.2 OvernightCap 3 Drops, 475000 5.7 1.1 Overnight

Experiment 2

A 10 microliter droplet of water containing Bacillus atrophaeus sporeswas dried onto the inner surface of the fitment from a ScholleClean-Clic® 4L (BG 0004 PM 900/8CC21 WB) bag-in-box bag. One milliliterof a performic acid mixture produced by a combination of water, formicacid, and hydrogen peroxide was placed in the bottom of the bag and thefitment then reinstalled; replicate inoculated fitments were placed onbags without the addition of performic solution in order to serve ascontrol samples. The bags were stored at ambient conditions(approximately 65-75° F.) for one week, the inoculation site on eachfitment was then swabbed, the swabs shaken in 3 mL of a saline solutionto harvest the recovered spores, and the harvest medium or its serialten-fold dilutions spread plated on tryptic soy agar medium andincubated for 48 hours at 35 C. The number of viable Bacillus atrophaeusspores recovered from untreated (Recovery Control) or performic treatedbags is shown in Table 2.

TABLE 2 The number of colony forming units (CFU) recovered from fitmentsof bags incubated without (Recovery Control) or with the addition of aperformic mixture. CFU Log CFU Log Sample Recovered Recovered ReductionRecovery Control 63800 4.8 NA Recovery Control 4300 4.6 NA Treated 0<0 >4.7 Treated 0 <0 >4.7

As illustrated by the data summarized in Table 2, no viable spores wererecovered from the performic treated bag sample fitments, while 46,000CFU or more of spores were recovered from the fitments on untreated,control bags.

Experiment 3

As in Experiment 2, a 10 microliter droplet of water containing Bacillusatrophaeus spores was dried on the inner surface of the fitment from aScholle Clean-Clic® 4L (BG 0004 PM 900/8CC21 WB) bag-in-box bag. Theinoculum level used was significantly greater than was used inExperiment 2. Referring now to FIGS. 1A, 1B, and 2. FIGS. 1A and 1B,which show inoculated fitments on which a 10 microliter water dropletcontaining greater than 10,000,000 (10 million) colony forming units ofBacillus atrophaeus spores was dried. The spore inoculation site on eachfitment was clearly visible after drying (FIGS. 1A and 1B); clearly thespot contains a very high concentration of spores. The area of the spotis about 0.4 cm² and it contains greater than 10,000,000 spores (a sporeconcentration greater than 25 million spores per square centimeter).This is a relatively high level of inoculation and the spore mass at theinoculation site is clearly visible. Referring to FIG. 2. The 10microliter water droplet dried onto the fitment shown in FIG. 1, viewedat about 25 times magnification using a stereomicroscope. Note the thickouter ring of spores formed as the droplet dried; a portion of thisthick outer ring has dislodged from the upper part of the dried dropletduring handling of the fitment before viewing. This outer ring containsa thick biomass of spores deposited one on top of the other many sporesdeep.

About one milliliter of a performic acid mixture produced by acombination of water, formic acid, and hydrogen peroxide was placed inthe bottom of the bag and the fitment then reinstalled; replicateinoculated fitments were placed on bags without the addition ofperformic solution in order to serve as control samples. The bags werestored at ambient conditions (approximately 65-75° F.) for 20, 44, and68 hours and the inoculation site on each fitment was then swabbed,recovered, and counted as in Experiment 2. The number of viable Bacillusatrophaeus spores recovered from untreated (Recovery Control) orperformic treated bags is shown in Table 3 below. Some surviving sporeswere recovered from one of the treated samples after 20 hours oftreatment (but not the replicate sample), but no viable spores wererecovered on any of the treated samples thereafter. About 7.5 logarithmcycles of viable spores (or more than 10 million CFU) were recoveredfrom the untreated, control samples.

TABLE 3 The number of colony forming units (CFU) recovered from fitmentsof bags incubated without (Recovery Control) or with the addition of 1mL of a performic mixture. Treated samples were assayed after 20, 44,and 68 hours; the control samples were assayed at 20 and 68 hours. CFULog CFU Log Sample Recovered Recovered Reduction Recovery Control34300000 7.5 NA Recovery Control 31400000 7.5 NA 20 Hrs 225 2.4 5.1 20Hrs 0 <0 >7.5 44 Hrs 0 <0 >7.5 44 Hrs 0 <0 >7.5 68 Hrs 0 <0 >7.5 68 Hrs0 <0 >7.5

Experiment 4

As in Experiments 2 and 3, a 10 microliter droplet of water containingBacillus atrophaeus spores was dried onto the inner surface of thefitment from a Scholle Clean-Clic® 4L (BG 0004 PM 900/8CC21 WB)bag-in-box bag. The inoculum level used was high as in Experiment 3. Fortreatment, 300 microliters or 100 microliters of a performic acidmixture produced by a combination of water, formic acid, and hydrogenperoxide was placed in the bottom of a bag and the fitment thenreinstalled; a replicate inoculated fitment was placed on a bag withoutthe addition of performic solution in order to serve as a controlsample. The bags were stored at ambient conditions (approximately 65-75°F.) for 50 hours and the inoculation site on each fitment was thenswabbed, recovered, and counted as in Experiments 2 and 3. The number ofviable Bacillus atrophaeus spores recovered from untreated (RecoveryControl) or performic treated bags is shown in Table 4 below. Nosurviving spores were recovered from either of the treated samples.About 7.7 logarithm cycles of viable spores (or more than 10 millionCFU) were recovered from the untreated, control sample.

After the 50 hour incubation at ambient conditions, sample bags treatedwith 100 microliters of the performic solution were filled with 3 litersof water, shaken, and then sampled and assayed for peroxide residualsusing CHEMetrics, Inc. (4295 Catlett Rd., Calverton, Va. 20138) ChemetsK-5510 assay procedures. The peroxide residuals were 1 part per million(ppm, mg/L) in the water sample. A peroxide residuals reading was alsotaken on a bag treated with 100 microliters of the performic solutionand assayed after 9 days of incubation at ambient conditions. Again thebag was filled with 3 liters of water, shaken, and then sampled andassayed as before. The peroxide residuals were now between 0.6 and 0.8ppm in the water sample.

TABLE 4 The number of colony forming units (CFU) recovered from fitmentsof bags incubated without (Recovery Control) or with the addition ofeither 300 microliters or 100 microliters of a performic mixture.Samples were assayed after 50 hours at room temperature. CFU Log CFU LogSample Recovered Recovered Reduction Recovery Control 54100000 7.7 NA300 microL, 50 Hrs 0 <0 >7.7 100 microL, 50 Hrs 0 <0 >7.7

Experiment 5

As in Experiments 2, 3, and 4, a 10 microliter droplet of watercontaining Bacillus atrophaeus spores was dried on the inner surface ofthe fitment from a Scholle Clean-Clic® 4L (BG 0004 PM 900/8CC21 WB)bag-in-box bag. The inoculum level used was a one-to-ten dilution ofthat used in Experiments 3 and 4. For treatment, 25 microliters of aperformic acid mixture produced by a combination of water, formic acid,and hydrogen peroxide was placed in the bottom of a bag and the fitmentthen reinstalled; a replicate inoculated fitment was placed on a bagwithout the addition of performic solution in order to serve as acontrol sample. The bags were stored at ambient conditions(approximately 65-75° F.) for 46 and 122 hours and the inoculation siteon each fitment was then swabbed, recovered, and counted as previouslydescribed. The number of viable Bacillus atrophaeus spores recoveredfrom untreated (Recovery Control) or performic treated bags is shown inTable 5 below. Surviving spore recovery from either of the treatedsamples was significantly less than that from the untreated, controlsamples.

After the 33 hours at ambient conditions incubation, a sample bagtreated with 25 microliters of the performic solution was filled with 3liters of water, shaken, and then sampled and assayed for peroxideresiduals using the Chemets K-5510 assay procedures. The oxidativeperoxide residuals were 0.3 parts per million (ppm) in the water sample.A second oxidative peroxide residuals reading was taken after 122 hoursof incubation at ambient conditions. Again the bag was filled with 3liters of water, shaken, and then sampled and assayed as before. Theoxidative peroxide residuals level was now 0.2 ppm in the water sample.The regulatory limit for oxidative peroxide residual levels under theseconditions is 0.5 ppm; clearly the disinfection process yielded peroxideoxidative levels well below the regulatory limit.

TABLE 5 The spore recovery from fitments of bags incubated without(Recovery Control) or with the addition of either 25 microliters of aperformic mixture. Samples were assayed after 46 and 122 hours at roomtemperature. CFU Log CFU Log Sample Recovered Recovered ReductionRecovery Control 2,600,000 6.4 NA Recovery Control 2,650,000 6.4 NA 25microL, 46 Hrs 2280 3.4 3.0 25 microL, 122 Hrs 8 0.9 5.5

Experiment 6

The effect of concentration of the added disinfection mixture wasinvestigated by comparing 500 microliter of the disinfection mixtureused in the previous demonstrations (call this mixture a 1×concentration of the disinfecting agent) and 200 microliters of amixture containing 3.8 times the level of peroxide as the 1×concentration mixture.

As in many of the previous exhibits, a 10 microliter droplet of watercontaining Bacillus atrophaeus spores was dried on the inner surface ofthe fitment from a Scholle Clean-Clic® 4L (BG 0004 PM 900/8CC21 WB)bag-in-box bag. For treatment, each of the two volumes and concentrationmixture combinations of water, formic acid, and hydrogen peroxide wereplaced in the bottom of a bag and the fitment then reinstalled; areplicate inoculated fitment was placed on a bag without the addition ofperformic solution in order to serve as a control sample.

The results obtained from this experiment are summarized in Table 6.These data illustrate that by elevating the concentration of thedisinfection mixture added to the package it is possible to acceleratethe migration/permeation of the disinfection mixture within the bag.

TABLE 6 The effect of the concentration of the disinfection mixture onthe time required to produce disinfection and sterilization within atreated bag. Untreated, Control 7.3 Logarithm Cycles of Colony FormingUnits Recovered 1X Concentration 500 μL 6.6 Logarithm Cycles of Colonyfor 17 Hours Forming Units Recovered 3.8X Concentration 3 Colony FormingUnits Recovered 200 μL for 17 Hours

Experiment 7

Demonstration tests were performed to document the ability of thismethod to effectively disinfect and sterilize even larger packages thanthe 4 liter bags employed previously. Fifty seven and nine-tenths (57.9)gallon bags were used for these tests (Scholle 200301 BG 0220 APHM800/800X). For these tests, a 10 microliter droplet of water containingBacillus atrophaeus spores was dried on the inner surface of the fitmentand also onto the inner surface of the bag immediately beneath thefitment.

Five milliliters or 2.5 mL of the disinfection mixture (1×concentration) was placed in the bag as low and as far below the fitmentas possible and the bag held upright and shaken slightly in order tocause the disinfection mixture to run to the bottom of the bag. One bagto which 5 mL of disinfection mixture was added and one bag to which 2.5mL was added were incubated at ambient temperatures in an unfolded flatposition, and one bag to which 5 mL of disinfection mixture was addedwas folded before incubation (see FIG. 3).

The bags were incubated for thirteen days and the results obtained forthe number of viable Bacillus atrophaeus spores recovered from anuntreated (Control) bag or the bags to which the disinfection mixturehad been added are summarized in Table 7 below.

TABLE 7 The numbers of viable colony forming units recovered from 57.9gallon drum bags. Innoculation Site Sample Fitment Bag Control 6.73 LogCFU 6.72 Log CFJ 5 mL of 1X   0 CFU   0 CFU Concentration 2.5 mL of 1X  0 CFU   0 CFU Concentration Folded Bag   0 CFU   0 CFU

Referring now to FIG. 3. For testing two 57.9 gallon drum bags wereincubated in a flat position or after careful folding (attempting toretain the added disinfection mixture in the bottom of the bag). Afterswab recovery of the Bacillus atrophaeus spores from the two inoculationsites on the disinfection mixture treated bags, the bags were filledwith 57.5 gallons (483 pounds) of water, mixed, and then sampled andassayed for peroxide residuals using the Chemets K-5510 assayprocedures. The oxidative peroxide residual determinations obtained areshown in Table 8.

TABLE 8 The oxidative residual levels obtained from 57.9 gallon drumbags after 13 days. 5 mL of 1X 0.3 mg/L Concentration 2.5 mL of 1X 0.1mg/L Concentration Folded Bag 0.4 mg/L (5 mL of 1X)

Experiment 8

This exhibit is an extension of the methods and results shown inExperiment 7 using the same 57.9 gallon drum bags used in Experiment 7(Scholle 200301 BG 0220 APHM 800/800X). The volumes and disinfectionmixture concentrations employed are shown in Table 9. The tested volumesand concentrations of the disinfection mixture were able to inactivateall the inoculated Bacillus atrophaeus spores after incubation for aboutfour (4) days at ambient conditions. Note that in these trials thedisinfection mixture was deposited at the foot end of the bag oppositethe fitment, the bag folded so as to retain the disinfection mixture atthat end of the bag during the folding process, and the bag was thenincubated in this folded position; the disinfection mixture wasnevertheless able to still migrate across the folding pattern toinactivate spores inoculated on the fitment. Greater than 6 (6.4)logarithm cycles of viable spore colony forming units were recoveredfrom the untreated, control bag.

Referring now to Table 9. In both trials the disinfection mixture wasdeposited at the foot end of the bag opposite the fitment, the bagfolded, and the bag treated in a folded position. Also, in theseparticular tests the oxidative potential of the residual contents of thebag after filling with 5.3 gallons of water were below the regulatorymaximum (regulatory limit is 0.5 mg/L) as the level of recoveredresiduals in these tests were 0.1 and 0.2 mg/L. These results show thatit is possible to sterilize these large bags (57.9 gallons) during roomtemperature storage in as short a time as four days and with negligibleand generally recognized as safe levels of residuals.

TABLE 9 Recovery of viable spores and oxidative residuals from 57.9gallon drum bags after about 4 days of ambient incubation. 95.5 Hrs 4 mL0 CFU Recovered Residuals Log Red Concentration 4.5 X 0 CFU Recovered0.1 mg/L >6.4 95 Hrs 6 mL 0 CFU Recovered Residuals Log Red 96 HrsControl 6.53 Log CFU Recovered 96 Hrs Control 6.32 Log CFU Recovered

Experiment 9

The previous exhibits employed bags with relatively high barrierproperties, i.e., the disinfectants and steriliants added were confinedto the bag, the materials and construction of the bags being relativelyinhibitory to the migration of moisture or gasses through the walls ofthe bag.

In this experiment, a polyolefin bag (Scholle 2.6 gallon 200119 BG 0010PP 900/1400 WB) with significantly lower barrier properties (a bagrelatively more permeable to moisture vapor or gasses) was employed todemonstrate the efficacy of the method even under such conditions andwith such materials wherein the treated container is not strictlyhermetic nor the treated volume strictly confined.

During initial trials, adding up to even 1 milliliter of thedisinfection mixture at the 1× concentration yielded no significantBacillus atrophaeus spore inactivation even after 55 hours of ambientincubation. Subsequent trials using 1.5 mL and 2.5 mL of the 1×concentration of the disinfection mixture yielded 2 logarithm cycles orless inactivation after 141 hours (>5.8 days) of ambient incubationusing a 6 logarithm cycle inoculation level of Bacillus atrophaeusspores.

After these poor initial results, a weight loss study was performed.Referring now to FIG. 4. The graph in FIG. 4 illustrates the loss ofweight when 1.5 mL of water, 1.5 mL of the disinfection mixture at the1× concentration, or 5 mL of the 1× concentration disinfection mixturewere sealed into the bag and incubated at ambient conditions inside asealed chamber containing Drierite (W. A. Hammond Drierite, Ltd, Xenia,Ohio). An approximately 0.013 to 0.015 grams per hour loss in weightfrom the bag was observed. The results obtained clearly demonstrate thatduring incubation, compounds in the bag are migrating through thepackaging material and being lost to the environment surrounding thebag.

The migration through the bag (effusion) necessitates an adjustment ofthe composition and amount of disinfection mixture needed to be added tothe bag in order to achieve sterilization. Follow on experimentsexamined the effects of using different concentrations and volumes ofthe disinfection mixture. As in previous experiments, a 10 microliterdroplet of water containing Bacillus atrophaeus spores was dried on theinner surface of the fitment from each of a duplicate series of bags. Ineach of the test bags, the volume of disinfection mixture at theconcentration listed in Table 10 was deposited near the center of thebag, the fitment replaced on each bag, and the bag incubated for 23 or25 hours at ambient temperatures (see Table 10), at which time theinoculation site on each fitment was swab recovered and viable sporesenumerated.

The results summarized in Table 10 illustrate that in these 2.6 gallonpolyolefin bags a range of disinfection mixture concentrations and addedvolumes were able to inactivate all or nearly all of the Bacillusatrophaeus spores within about a day of incubation at ambienttemperatures and conditions. These results clearly demonstrate that anappropriate volume and concentration of the disinfection mixture can beused to disinfect and sterilize the internal volume of polyolefin bags.

TABLE 10 The disinfection and sterilization effects of using variousvolume and concentration combinations to treat 2.6 gallon polyolefinbags. (Log Red is a convenient abbreviation for Logarithm CycleReduction in Spore Viability). 25 Hrs 200 μL   0 CFU Recovered ResidualsLog Red Concentration 2.7 X 0.15 mg/L >7.31 23 Hrs 200 μL   0 CFURecovered Residuals Log Red Concentration 4.5 X  0.3 mg/L >7.31 25 Hrs150 μL 6.64 Logs Recovered Residuals Log Red Concentration 1.3 X 0.05mg/L   0.66 23 Hrs 150 μL   0 CFU Recovered Residuals Log RedConcentration 2.2 X  0.0 mg/L >7.31

Experiment 10

This exhibit is an extension of the methods and results shown inExperiment 9 to a second, larger a polyolefin bag (Scholle 200233 BG0020 PP 900/1400). The volumes and disinfection mixture concentrationsemployed are shown in Table 11. Again it was seen that the selectedvolume and concentration of the disinfection mixture was able toinactivate all the inoculated Bacillus atrophaeus spores after one dayof ambient incubation. Note that in one trial the disinfection mixturewas deposited at the foot end of the bag opposite the fitment, the bagfolded so as to retain the disinfection mixture at that end of the bagduring the folding process, and then the bag incubated in this foldedposition; the disinfection mixture was nevertheless still able tomigrate (across the folding pattern) to inactivate spores inoculated onthe fitment. Greater than 7 (7.14) logarithm cycles of viable sporecolony forming units were recovered from the untreated, control bag.

Referring now to Table 11. In these particular tests the oxidativepotential of the residual contents of the bag after filling with 5.3gallons of water were about twice the allowable regulatory maximum(regulatory limit is 0.5 mg/L; recovered residual levels in these testswere 1.0 mg/L).

TABLE 11 The disinfection and sterilization effects of using variousvolume and concentration combinations to treat 5.3 gallon polyolefinbags. 24 Hours 400 mL 0 CFU Recovered Residuals Log Red Concentration4.5X 0.1 mg/L  .7.14 24 Hours Repeat 400 mL 0 CFU Recovered ResidualsLog Red Concentration 4.5X 0.1 mg/L >7.14 24 Hours Folded 400 mL 0 CFURecovered Residuals Log Red Concentration 4.5X Folded 0.1 mg/L >7.14

While the novel technology has been illustrated and described in detailin the figures and foregoing description, the same is to be consideredas illustrative and not restrictive in character, it being understoodthat only the preferred embodiments have been shown and described, andthat all changes and modifications that come within the spirit of thenovel technology are desired to be protected. As well, while the noveltechnology was illustrated using specific examples, theoreticalarguments, accounts, and illustrations, these illustrations and theaccompanying discussion should by no means be interpreted as limitingthe technology. All patents, patent applications, and references totexts, scientific treatises, publications, and the like referenced inthis application are incorporated herein by reference in their entirety.

1. A method for controlling contamination within a partitioned orotherwise defined space, comprising: contacting surfaces within apartitioned or otherwise defined space with a chemical in the vaporphase, wherein the chemical in the vapor phase is selected from thegroup consisting of: peroxides, peracids, or moieties that include atleast one peroxide bond, wherein the surface is included in apartitioned or otherwise defined space and wherein the surface isexposed to the chemical in its vapor phase for at least 5 minutes at atemperature below 50 C.
 2. The method according to claim 1, wherein thesurface is part of an article in a container selected from groupconsisting of: packaging, containers, bags, pouches, cans, tins,buckets, tubs, bottles, ampoules, barrels, sleeves, capsules, boxes,other formed packages or structures.
 3. The method according to claim 2,wherein the containers are flexible.
 4. The method according to claim 2,wherein the containers are rigid.
 5. The method according to claim 2,wherein the containers further include at least one accessory selectedfrom the group consisting of: fitments, valves, bungs, lids, vents,ports, spouts, drains, covers and caps, seals, or membranes.
 6. Themethod according to claim 1, wherein the time of incubation of theperoxide, peracid, or a mixture of compounds, one or more of whichincludes a peroxide bond in the partitioned or defined space is longerthan about five minutes.
 7. The method according to claim 1, wherein thetemperature of incubation of the peroxide, peracid, or a mixture ofcompounds, one or more of which includes a peroxide bond in thepartitioned or defined space is equal to or less than about 50 C.
 8. Themethod according to claim 1, wherein the peroxide concentration added toor used in the partitioned or defined space to form a peracid or amixture of compounds one or more of which includes a peroxide bond, isequal to or greater than about 2%.
 9. The method according to claim 1,wherein the peroxide mixture added to or used in the partitioned ordefined space includes formic acid (methanoic acid) or performic acid(permethanoic acid) either of which is added or present at aconcentration greater than or equal to about 2%.
 10. The methodaccording to claim 1, wherein the peroxide mixture added to or used inthe partitioned or defined space includes peracetic acid (ethanoic acid)or peracetic acid (perethanoic acid), or propionoic acid (propanoicacid) or propionoic peracid (propanoic peracid), or a mixture of thesewith or without the addition of formic acid, any of or the combinationof which is added or present at a concentration greater than or equal to2%.
 11. The method according to claim 1, wherein the peroxide mixtureadded to or used in the partitioned or defined space includes at thetime of addition, or subsequently evolves to form a mixture that isgenerally recognized as safe for consumption or other usage.
 12. Amethod for reducing the number of biological contaminants in apartitioned or otherwise defined space or volume, comprising the stepsof: mixing formic acid, hydrogen peroxide and water with or without theaddition of other compounds; wherein the mixture produces performic acidin situ; and contacting at least one surface with said performic acidformed in situ wherein the surface is within a partitioned or otherwisedefined space or volume.
 13. The method according to claim 12, whereinthe space or volume is the interior of a container.
 14. The methodaccording to claim 13, wherein the container is selected from the groupconsisting of packaging, bags, pouches, cans, tins, boxes, buckets,tubs, bottles, ampoules, barrels, sleeves, formed packages or structuresor the like.
 15. The method according to claim 12, wherein thepartitioned or otherwise defined space or volume includes at least onearticle and the article is present in the space or volume at the sametime as a microbiocidal concentration of peracid or peroxide bondcontaining vapors.
 16. The method according to claim 13, wherein theinterior of the container includes at least one article and the articleis inside the container at the same time as a microbiocidalconcentration of peracid or peroxide bond containing vapors.
 17. Themethod according to claim 12, wherein the peracid or peroxide bondincluding moieties that diminish over time and the residue from peracidor peroxide bond including mixture is too low to pose an unacceptablethreat to human or animal health.
 18. A system for controllingcontamination within a partitioned or otherwise defined space or volume,comprising: producing a vapor that includes a microbiocidalconcentration of peracid or peroxide bonds within a partitioned orotherwise defined space or volume, wherein the concentration of vaporsis such that at least about 5 minutes of contact between the vapor andan interior surface of the space or volume is required to inactivate atleast about 90 percent of the microbes within the space or volume. 19.The system according to claim 18, wherein said vapor is formed by amixture that includes peracid or peroxide formic acid (methanoic acid)or performic acid (permethanoic acid) either of which is added orpresent at a concentration greater than or equal to about 2%.
 20. Thesystem according to claim 18, wherein partitioned or otherwise definedspace or volume is within a container.